Microwave-assisted peptide synthesis

ABSTRACT

An instrument and method for accelerating the solid phase synthesis of peptides is disclosed. The method includes the steps of deprotecting a protected first amino acid linked to a solid phase resin by admixing the protected linked acid with a deprotecting solution in a microwave transparent vessel while irradiating the admixed acid and solution with microwaves, then activating a second amino acid by adding the second acid and an activating solution to the same vessel while irradiating the vessel with microwaves, then coupling the second amino acid to the first acid while irradiating the composition in the same vessel with microwaves, and cleaving the linked peptide from the solid phase resin by admixing the linked peptide with a cleaving composition in the same vessel while irradiating the composition with microwaves.

RELATED APPLICATIONS

This is a continuation of Ser. No. 10/604,022 filed Jun. 23, 2003 andnow U.S. Pat. No. 7,393,920.

BACKGROUND OF THE INVENTION

This application incorporates by reference the sequence listingsubmitted concurrently herewith on paper. This paper copy of thesequence listing is entitled “Sequence Listing.”

The present invention relates to solid-phase peptide synthesis (SPPS),and in particular relates to microwave-assisted techniques for SPPS.

The early part of the twentieth century saw the birth of a novel conceptin scientific research in that synthetically produced peptides couldgreatly facilitate the study of the relationship between chemicalstructure and biological activity. Until that time, the study ofstructure-activity relationships between peptides and their biologicalfunction had been carried out using purified, naturally occurringpeptides. Such early, solution-based techniques for peptide purificationwere plagued with problems, however, such as low product yield,contamination with impurities, their labor-intensive nature and theunpredictable solubility characteristics of some peptides. During thefirst half of the twentieth century some solution-based synthesistechniques were able to produce certain “difficult” peptides, but onlyby pushing known techniques to their limits. The increasing demand forhigher peptide yield and purity resulted in a breakthrough techniquefirst presented in 1963 for synthesizing peptides directly from aminoacids, now referred to as solid-phase peptide synthesis (SPPS).

The drawbacks inherent in solution-based peptide synthesis have resultedin the near-exclusive use of SPPS for peptide synthesis. Solid phasecoupling offers a greater ease of reagent separation, eliminates theloss of product due to conventional chemistries (evaporation,recrystallization, etc.), and allows for the forced completion of thereactions by adding excess reagents.

Peptides are defined as small proteins of two or more amino acids linkedby the carboxyl group of one to the amino group of another. Accordingly,at its basic level, peptide synthesis of whatever type comprises therepeated steps of adding amino acid molecules to one another or to anexisting peptide chain of acids.

The synthetic production of peptides is an immeasurably valuable tool inthe field of scientific research for many reasons. For example, someantiviral vaccines that exist for influenza and the humanimmunodeficiency virus (HIV) are peptide-based. Likewise, some work hasbeen done with antibacterial peptide-based vaccines (diphtheria andcholera toxins). Synthetically altered peptides can be labeled withtracers, such as radioactive isotopes, and used to elucidate thequantity, location, and mechanism of action of the native peptide'sbiological acceptor (known as a receptor). This information can then beused to design better drugs that act through that receptor. Peptides canalso be used for antigenic purposes, such as peptide-based antibodies toidentify the protein of a newly discovered gene. Finally, some peptidesmay be causative agents of disease. For example, an error in thebiological processing of the beta-amyloid protein leads to the“tangling” of neuron fibers in the brain, forming neuritic plaques. Thepresence of these plaques is a pathologic hallmark of Alzheimer'sDisease. Synthetic production of the precursor, or parent molecule, ofbeta-amyloid facilitates the study of Alzheimer's Disease.

These are, of course, only a few of the wide variety of topics andinvestigative bases that make peptide synthesis a fundamental scientifictool.

The basic principle for SPPS is the stepwise addition of amino acids toa growing polypeptide chain that is anchored via a linker molecule to asolid phase particle which allows for cleavage and purification once thecoupling phase is complete. Briefly, a solid phase resin support and astarting amino acid are attached to one another via a linker molecule.Such resin-linker-acid matrices are commercially available (e.g.,Calbiochem, a brand of EMD Biosciences, an affiliate of Merck KGaA ofDarmstadt, Germany; or ORPEGEN Pharma of Heidelberg, Germany, forexample). The starting amino acid is protected by a chemical group atits amino terminus, and may also have a chemical side-chain protectinggroup. The protecting groups prevent undesired or deleterious reactionsfrom taking place at the alpha-amino group during the formation of a newpeptide bond between the unprotected carboxyl group of the free aminoacid and the deprotected alpha-amino of the growing peptide chain. Aseries of chemical steps subsequently deprotect the amino acid andprepare the next amino acid in the chain for coupling to the last.Stated differently, “protecting” an acid prevents undesired side orcompeting reactions, and “deprotecting” an acid makes its functionalgroup(s) available for the desired reaction.

When the desired sequence of amino acids is achieved, the peptide iscleaved from the solid phase support at the linker molecule. Thistechnique consists of many repetitive steps making automation attractivewhenever possible.

Many choices exist for the various steps of SPPS, beginning with thetype of reaction. SPPS may be carried out using a continuous flow methodor a batch flow method. Continuous flow is useful because it permitsreal-time monitoring of reaction progress via a spectrophotometer.However, continuous flow has two distinct disadvantages in that thereagents in contact with the peptide on the resin are diluted, and scaleis more limited due to physical size constraints of the solid phaseresin. Batch flow occurs in a filter reaction vessel and is usefulbecause reactants are accessible and can be added manually orautomatically.

Other choices exist for chemically protecting the alpha-amino terminus.A first is known as “Boc” (N{acute over (α)}-t-butoxycarbonyl). Althoughreagents for the Boc method are relatively inexpensive, they are highlycorrosive and require expensive equipment. The preferred alternative isthe “Fmoc” (9-fluorenylmethyloxycarbonyl) protection scheme, which usesless corrosive, although more expensive, reagents.

For SPPS, solid support phases are usually polystyrene suspensions; morerecently polymer supports such as polyamide have also been used.Preparation of the solid phase support includes “solvating” it in anappropriate solvent (dimethyl formamide, or DMF, for example). The solidphase support tends to swell considerably in volume during salvation,which increases the surface area available to carry out peptidesynthesis. As mentioned previously, a linker molecule connects the aminoacid chain to the solid phase resin. Linker molecules are designed suchthat eventual cleavage provides either a free acid or amide at thecarboxyl terminus. Linkers are not resin-specific, and include peptideacids such as 4-hydroxymethylphenoxyacetyl-4′-methylbenzyhydrylamine(HMP), or peptide amides such as benzhydrylamine derivatives.

Following the preparation of the solid phase support with an appropriatesolvent, the next step is to deprotect the amino acid to be attached tothe peptide chain. Deprotection is carried out with a mild basetreatment (picrodine or piperidine, for example) for temporaryprotective groups, while permanent side-chain protecting groups areremoved by moderate acidolysis (trifluoroacetic acid, or TFA, as anexample).

Following deprotection, the amino acid chain extension, or coupling, ischaracterized by the formation of peptide bonds. This process requiresactivation of the C-alpha-carboxyl group, which may be accomplishedusing one of five different techniques. These are, in no particularorder, in situ reagents, preformed symmetrical anhydrides, activeesters, acid halides, and urethane-protected N-carboxyanhydrides. The insitu method allows concurrent activation and coupling; the most populartype of coupling reagent is a carbodiimide derivative, such asN,N′-dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide.

After the desired sequence has been synthesized, the peptide is cleavedfrom the resin. This process depends on the sensitivity of the aminoacid composition of the peptide and the side-chain protector groups.Generally, however, cleavage is carried out in an environment containinga plurality of scavenging agents to quench the reactive carbonium ionsthat originate from the protective groups and linkers. One commoncleaving agent is TFA.

In short summary SPPS requires the repetitive steps of deprotecting,activating, and coupling to add each acid, followed by the final step ofcleavage to separate the completed peptide from the original solidsupport.

Two distinct disadvantages exist with respect to current SPPStechnology. The first is the length of time necessary to synthesize agiven peptide. Deprotection steps can take 30 minutes or more. Couplingeach amino acid to the chain as described above requires about 45minutes, the activation steps for each acid requires 15-20 minutes, andcleavage steps require two to four hours. Thus, synthesis of a meretwelve amino acid peptide may take up to 14 hours. To address this,alternative methods of peptide synthesis and coupling have beenattempted using microwave technology. Microwave heating can beadvantageous in a large variety of chemical reactions, including organicsynthesis because microwaves tend to interact immediately and directlywith compositions or solvents. Early workers reported simple couplingsteps (but not full peptide synthesis) in a kitchen-type microwave oven.Such results are not easily reproducible, however, because of thelimitations of a domestic microwave oven as a radiation source, a lackof power control, and reproducibility problems from oven to oven. Othershave reported enhanced coupling rates using microwaves, but haveconcurrently generated high temperatures that tend to cause the solidphase support and the reaction mixtures to degenerate. Sample transferbetween steps has also presented a disadvantage.

Another problem with the current technology is aggregation of thepeptide sequence. Aggregation refers to the tendency of a growingpeptide to fold back onto itself and form a loop, attaching via hydrogenbonding. This creates obvious problems with further chain extension.Theoretically, higher temperatures can reduce hydrogen bonding and thusreduce the fold-back problem, but such high temperatures can createtheir own disadvantages because they can negatively affectheat-sensitive peptide coupling reagents. For this reason, SPPSreactions are generally carried out at room temperature, leading totheir characteristic extended reaction times.

SUMMARY OF THE INVENTION

In one aspect, the invention is a process for the solid phase synthesisof peptides, which comprises the steps of: (a) deprotecting a firstamino acid linked to a solid phase resin by removing protective firstchemical groups; (b) activating chemical groups on a second amino acidto prepare the second amino acid for coupling with the first amino acid;(c) coupling the activated second amino acid to the deprotected firstamino acid to form a peptide from the first and second amino acids; and(e) applying microwave energy to accelerate the deprotecting,activating, and coupling cycle.

In another aspect the invention is an apparatus for the acceleratedsynthesis of peptides by the solid phase method, that comprises areaction cell that is transparent to microwave radiation; a passagewayfor adding liquids to the reaction cell; a passageway for removingliquids but not solids from the reaction cell; a microwave cavity forholding the cell; and a microwave source in wave communication with thecavity.

In yet another aspect, the invention is a vessel system for theaccelerated synthesis of peptides by the solid phase method, the vesselsystem comprising: a reaction cell that is transparent to microwaveradiation; a first passageway in fluid communication with the cell fortransferring solid phase resin between a resin source external to thecell and the cell; a second passageway in fluid communication between atleast one amino acid source and the cell for adding amino acids to thecell; a third passageway in gaseous communication with an inert gassource and with a vent for applying gas pressure to and releasing gaspressure from the cell so that the controlled flow of gases to and fromthe cell can be used to add and remove fluids and flowing solids to andfrom the cell.

In yet another aspect the invention is a process for accelerating thesolid phase synthesis of peptides, and comprising: deprotecting aprotected first amino acid linked to a solid phase resin by admixing theprotected linked acid with a deprotecting solution in a microwavetransparent vessel while irradiating the admixed acid and solution withmicrowaves; activating a second amino acid by adding the second acid andan activating solution to the same vessel while irradiating the vesselwith microwaves; coupling the second amino acid to the first acid whileirradiating the composition in the same vessel with microwaves; andcleaving the linked peptide from the solid phase resin by admixing thelinked peptide with a cleaving composition in the same vessel whileirradiating the composition with microwaves.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating certain aspects of solidphase peptide synthesis.

FIG. 2 is a perspective view of a synthesis instrument according to thepresent invention.

FIGS. 3, 4 and 5 are perspective views of a reaction vessel and adapteraccording to the present invention.

FIG. 6 is a flow circuit diagram illustrating aspects of the presentinvention.

FIG. 7 is a cut-away perspective view of the cavity and waveguide of thepresent invention.

FIG. 8 is the mass spectrum of one peptide synthesized according to themethod of the invention.

FIG. 9 is the mass spectrum of a second peptide synthesized according tothe method of the invention.

DETAILED DESCRIPTION

The invention is an apparatus and method for the solid phase synthesisof one or more peptides, specifically utilizing microwave energy toaccelerate the method.

FIG. 1 is a schematic diagram illustrating some aspects of the solidphase peptide synthesis process. It will be understood that FIG. 1 isgeneral in nature and is not limiting of the invention. FIG. 1illustrates a first amino acid 10 that includes an N-alpha protectivegroup 11 and a side chain protective group 12 attached to it. A linkingmolecule 13 is attached to a resin support 14. In a first stepdesignated by the arrow 15, the first acid and its protective groups 11and 12 are attached to the linker 13 and the resin support 14. In asecond step indicated by the arrow 17, the N-alpha protective group isremoved (“deprotected”) to produce the structure in which the first acid10 and its side chain-protecting group 12 are linked to the support 14through the linker molecule 13. In the next step, indicated by the arrow21, the first amino acid 10 is coupled to a second amino acid designatedat 20, which similarly has an N-alpha protective group 11 and anactivation group 22 attached to it to encourage the coupling. Followingthe coupling step 21, the resulting structure includes the first acid 10and the second acid 20 connected to one another and still including theN-alpha protective group 11 attached to the second acid 20 and the sidechain protective group 12 attached to the first acid 10 with theconnected acids being in turn linked to the support 14 through thelinking molecule 13. Additional acids, represented by the brokenrectangle 25 are added in the same manner (arrow 21′) to lengthen thepeptide chain as desired.

In the final step, the connected acids 10, 20 and 25 are cleaved,represented by the arrow 23, from the protective groups and the supportto result in the desired peptide separate from the resin support 14. Thecoupling steps can, as indicated a number of times elsewhere herein, berepeated as many times as desired to produce a resulting peptide.

FIG. 2 illustrates one commercial embodiment of the present inventionbroadly designated at 30. FIG. 2 illustrates some of the broadstructural aspects of the invention, the details of which will beexplained with respect to FIGS. 3 and 6.

First, FIG. 2 illustrates the microwave portion of the device 31. Theportion of the instrument that applies microwave irradiation to thevessel is preferably a single-mode cavity instrument that can becontrolled to apply suitable amounts of power to the sample sizes andmaterials used in the method of the invention. In the preferredembodiment of the invention, the microwave portion of the instrument hasthe design and operation that is set forth in a number of co-pending andcommonly assigned U.S. patents and applications. These include U.S.published applications Nos. 20030089706, 20020117498, 20030199099 and20040020923; and U.S. Pat. No. 6,744,024. The disclosures of all ofthese references are incorporated entirely herein by reference.Commercial versions of such single-mode microwave instruments areavailable from the assignee of the present invention, CEM Corporation,of Matthews, N.C., under the DISCOVERY™, VOYAGER™, and EXPLORER™ tradenames.

With those considerations in mind, FIG. 2 illustrates the location ofthe cavity 32, the housing 33, and an appropriate display 34, forproviding instructions or information during operation. A plurality ofamino acid source containers or bottles are each respectively indicatedat 35. The respective resin containers are illustrated at 36, and theproduct peptide containers are designated at 37. A series of fluidpassageways are illustrated by the portions of tubing broadly designatedat 40 and will be discussed in more detail with respect to FIG. 6.Similarly, the instrument 30 includes an upper housing portion 41, whichincludes an appropriate manifold, for physically transporting the fluidsand resins in the manner described herein. Although the manifold is notillustrated, it can comprise any series of passageways and valves thatserve to direct the fluids in the manner described herein andparticularly described with respect to the circuit diagram of FIG. 6.

Thus, in the embodiment illustrated in FIG. 2, up to 20 different aminoacids can be incorporated in the respective containers 35, and up to 12different peptides can be produced and placed in the respectivecontainers 37 in automated fashion. It will be understood that these arecommercial embodiment numbers, however, and that the invention isneither limited to this number nor does it need to have as many sourcesor product containers as are illustrated.

FIG. 2 also illustrates a complimentary series of passageways shown asthe tubing broadly designated at 42 that are immediately connected tothe reaction vessel adapter 43, which is partially illustrated in FIG.2, but is described in more detail with respect to FIGS. 3, 4, and 5.

FIG. 3 is a partial perspective view of the reaction vessel 45 and thevessel adapter 43, portions of which were also illustrated in FIG. 2.The reaction vessel 45 is preferably pear-shaped and formed of amaterial that is transparent to microwave radiation. Preferred materialsinclude, but are not limited to, glass, Teflon, and polypropylene. Afirst passageway, shown as the tubing 46, is in fluid communication withthe reaction vessel (or “cell,” the terms are used interchangeableherein) 45 for transferring solid phase resin between a resin sourceexternal to the cell 45 and the cell 45. A second passageway 47 is influid communication between at least one amino acid source (FIG. 6) andthe cell 45 for adding amino acids to the cell 45. A third passageway 50is in gas communication with an inert gas source (FIG. 6) and with avent (FIG. 6) for applying and releasing gas pressure to and from thecell 45, so that the controlled flow of gas in the manifold and to andfrom the cell 45 can be used to add and remove fluids and flowing solidsto and from the cell 45.

FIG. 3 also illustrates that the second passageway 47 also includes afilter, shown as the frit 51, for preventing solid-phase resin fromentering the second passageway 47 from the cell 45.

In preferred embodiments, the invention further comprises a fourthpassageway 52, in fluid communication between an external solvent source(FIG. 6) and the cell 45 for flushing the cell 45 with solvent. Asillustrated in FIG. 3, the fourth passageway 52 includes a spray head 53or equivalent structure for adding the solvent to the cell 45.

The adapter 43 is formed of a microwave transparent and chemically inertmaterial, which is preferably formed of a polymer, such as a fluorinatedpolymer (e.g., PTFE) or an appropriate grade of polypropylene. Theadapter 43 is preferably a solid cylinder with the passageways 46, 47,50, and 52 drilled or bored there through. The passageways 46, 47, 50,52 can simply comprise the bore holes through the adapter 43, butpreferably may also include tubing, which again is formed of a microwavetransparent, chemically inert material such as PTFE, PTFE variations, orpolypropylene. The tubing is preferably ⅛ inch outside diameter and 1/16inch inside diameter.

Although not illustrated in FIG. 3 (to reduce the complexity of thedrawing), the vessel neck 54 preferably is externally threaded andengages an internally threaded bore hole 55 in lower portions of theadapter 43. The threaded engagement between the vessel 45 and theadapter 43 permits secure engagement between these two items, and alsopermits the vessel 45 to be easily engaged and disengaged to and fromthe adapter 43. In particular, differently sized vessels or vesselsformed of different materials can be substituted and still fit theadapter 43, provided the necks are of the same size and threading.

As some final details, FIG. 3 also includes threaded fittings 56, 57,60, and 62 to the respective first, second, third and fourth passageways46, 47, 50 and 52. These permit the entire adapter 43 and vessel 45 tobe easily connected to and removed from the remainder of the instrument30.

FIGS. 4 and 5 are respective assembled and exploded perspective drawingsof the adapter of FIG. 3, and thus illustrate the same elements. Bothfigures include the adapter 43 and the cell 45. The threaded fittings57, 60, 56, and 62 are visible in FIG. 5, with 57, 60 and 56 alsovisible in 54. The exploded view of FIG. 5 also illustrates portions ofthe first and second passageways, 46, 47, as well as the threaded vesselneck 54 and the board opening 55 in the lower portions of the adapter43.

FIG. 6 is a flow circuit diagram for the present invention. Whereverpossible, the elements illustrated in FIG. 6 will carry the samereference numerals as in the other drawings. Because most of theelements symbolized in FIG. 6 are commonly available and wellunderstood, they will not be described in particular detail, as those ofskill in this art can practice the invention based on FIG. 6 withoutundue experimentation.

Accordingly, FIG. 6 illustrates a vessel system for the acceleratedsynthesis of peptides by the solid-phase method. The vessel systemcomprises the reaction cell (or vessel) 45, which is indicated in FIG. 6schematically as a square. Otherwise, the reaction cell 45 has all ofthe characteristics already described and which will not be repeatedwith respect to FIG. 6. The first passageway 46 is in fluidcommunication with the cell 45 for transferring solid phase resinbetween an external resin source 36 and the cell 45. Three resinsources, 36 (A, B and C) are illustrated in FIG. 6 and correspond to theresin sources 36 illustrated in FIG. 2. As set forth with respect toFIG. 2, the number of resin sources is elective rather than mandatorywith 12 being shown in the embodiment of FIG. 2, and three illustratedin FIG. 6 for purposes of simplicity and schematic understanding. Eachof the resin sources 36 is in communication with a respective three-wayvalve 64, A, B and C, and in turn, to an appropriate resin line 65, A, Band C and then another three-way valve 66 adjacent to cell 45 fordelivering resin through the first passageway 46 into the cell 45. Thethree-way valve 66 is immediately in communication with anotherthree-way valve 67, the purpose of which will be described shortly.

FIG. 6 also shows the second passageway 47, which is in communicationwith at least one of the amino acid sources 35, which are illustratedagain as rectangles in the upper portions of FIG. 6. The schematicallyillustrated amino acid sources or containers 35 correspond to thecontainers 35 illustrated in FIG. 2.

The third passageway 50 is in gas communication with an inert gas source70 and with a vent 71 for applying gas pressure to and releasing gaspressure from the cell 45, so that the controlled flow of gasses to andfrom the cell 45 can be used to add and remove fluids and flowing solidsto and from the cell. The third passageway 50 accomplishes this inconjunction with at least one valve 72 which, depending upon itsorientation, permits the third passageway 50 to communicate with eitherthe gas source 70 or the vent 71. The gas source can be any gas that canappropriately be pressurized and that does not otherwise interfere withthe chemistry of the peptide synthesis or the elements of the instrumentitself. Thus, a number of inert gases are suitable, with pressurizednitrogen being typically favored for reasons of wide availability, lowercost, ease of use, and lack of toxicity. FIG. 6 illustrates that thenitrogen supply 70 is controlled through a two-way valve 72 and anappropriate regulator 73, which also may include a filter. In theorientation of FIG. 6, the gas line from the two-way valve 72 to thevent 71 is labeled at 74, and the passageway from the valve 72 to theregulator 73 is designated at 75.

FIG. 6 also illustrates the filter 51 in the second passageway 47 forpreventing the solid phase resin from entering the second passagewayfrom the cell 45.

FIG. 6 also illustrates the fourth passageway 52 along with the sprayhead 53. As described with respect to FIG. 3, the fourth passageway 52is in fluid communication with one or more external solvent sourcesthree of which are illustrated at 76, A, B and C. Two other externalsolvent sources 77 and 80 are separately labeled because of theiroptionally different fluid paths.

FIG. 6 also illustrates the manner in which the pressurized gas from thesource 70 can be used to both deliver compositions to, and then removethem from, the reaction cell 45 as desired whether they be peptides,solvent, wastes, or resin. Thus, in one aspect of such delivery, FIG. 6illustrates a gas passage 81 that communicates with several items.First, the gas passageway 81 communicates with a series of two-wayvalves designated at 82A, B, C and D that each provide a gas passagewhen the respective valve is open to its corresponding amino acidcontainer 35. Pressurized gas entering a container 35 pushes the acidthrough the respective delivery lines 83A, B, C or D, which in turncommunicates with a respective acid valves 84 A, B, C and D and thenwith the second passageway 47 and its respective two-way valve 85 andthree-way valve 86. To illustrate, when valves 82A and 84A are open, andthe remaining valves 82B, C and D are closed, gas from the source 70 canbe directed through the gas passage 81, through valve 82A, into aminoacid bottle 35A, from the bottle 35A through the valves 84A, 85 and 86,and then into the cell 45.

The respective valves are automated in order to provide the cell withthe desired composition (e.g. resin, solvent, acid) at the appropriatepoint in the synthesis, as well as to remove compositions from the cell(peptides, waste) at other appropriate points. The required programmingand processor capacity is well within the capability of a personalcomputer-type processor (e.g. PENTIUM III®), and the use of automatedcontrols and sequences is generally well understood in this and relatedarts, e.g. Dorf, The Electrical Engineering Handbook, 2d Ed. (CRC Press1997).

It should be understood that while many amino acids exist, the twentysource containers of this apparatus are intended, but not limited to,contain the twenty “common” amino acids for synthesizing proteins thatare well known to those skilled in this art. These commerciallyavailable common amino acids can be purchased in chemically “protected”form (also from Sigma-Aldrich) to prevent unwanted and/or deleteriousreactions from occurring.

Solvent can be delivered to the cell in an analogous manner. Thesolvents communicate with the gas passage 81 through the valves 87A, Band C and 90 and 91. This places the gas in direct communication withthe external solvent tanks 76A, B and C and 77 and 80. External solventtanks 76A, B and C are further in communication with respective two-wayvalves 92A, B and C and respective three-way valves 93 and 94. These alllead, when the valves are appropriately oriented, to the secondpassageway 47 for delivering solvent to the reaction vessel 45 using gaspressure in the same manner that the acids are delivered. A TFA solventis used in external reservoirs 76C and thus can be directed throughalternative lines for optional isolation.

FIG. 6 also indicates that the gas source 70 can be used to drive itemsfrom the cell 45 directly by closing all of the valves to the aminoacids and the external solvent reservoirs, and then directing the gasthrough the regulator and filter 92 and its associated passageway 93directly to valves 67 and 66 and then into the first passageway 46 andthe cell 45.

Alternatively, the first passageway 46 can be used to empty the cell 45.In this aspect, valve 72 is set to direct gas from the source 70 andthrough the passage 75 to the valve 72 and through the third passageway50 and into the cell. The gas pressure then directs fluids in the cell45 through either second passageway 47 or first passageway 46 dependingupon the orientation of the valves 86, 66 and 67. FIG. 6 alsoillustrates an additional three-way valve 95 that can direct product tothe product containers 37A, B and C, which correspond to the productcontainers 37 illustrated in FIG. 2. An appropriate set of productvalves 96A, B and C can be opened or closed as desired to direct thedesired peptide product to the desired product container 37A, B or C.

Alternatively, depending upon the orientation of valves 86, 66, 67 and95, and together with additional two-way valve 100 and three-way valve101 adjacent to waste containers 102A and 102B, materials can bedirected from the cell 45 to either of the waste containers 102A and B.

The pressurized gas from the source 70 can also be used to deliverresin. In this aspect, the pressurized gas is directed through the gaspassage 81 and through the three-way valves 103 and 104. With respect todelivery of resin, however, when both of the valves 103 and 104 are opento the resin containers, they direct the pressurized gas to threerespective valves 105A, B and C which in turn are in communication withthe resin containers 36 and the exit valves 64A, 64B and 64C which thenuse the gas pressure delivered to force the resin through the resin line65 and eventually to the first passage 46 for delivery into the reactionvessel 45.

The resin sources may contain variable amounts and kinds of resins,including, but not limited to, Wang resins, Trityl resins, and Rink acidlabile resins; the resins are commercially available from vendors suchas Sigma-Aldrich Corp., Saint Louis, Mo. 63101.

Solvent can be directed to the resin containers 36A, B, C, from theexternal reservoirs 77, 80 using the valves 103, 104 between the solventreservoirs and the resin containers.

FIG. 6A is a more detailed illustration of the valving system adjacentthe reaction vessel 45. In particular, FIG. 6A shows a series of liquidsensors 106, 107 and 110 in conjunction with a series of three-wayvalves 111, 112, 113, 114 and 115. The operation of the valves inaccordance with the sensors permits a metered amount of liquid to beadded to the reaction vessel 45 as may be desired or necessary. Forexample, with the valves 111, 113 and 114 shown in the orientation ofFIG. 6A, fluid can flow directly from valve 86 all the way to thoseportions of second passageway 47 that extend immediately into thereaction vessel 45. Alternatively, if valve 111 is open towards valve112, liquid will flow through valves 111 and 112 until it reaches theliquid sensors 107 and 110. The liquid sensors inform the system when aproper or desired amount of liquid is included, which can then bedelivered by changing the operation of valve 112 to deliver to the valve113, and then to the valve 114, and then to the second passageway 47 andfinally into the cell 45 as desired.

Thus, in overall fashion, FIG. 6 illustrates the delivery of precursorcompositions (amino acids, solvents, resin, deprotectants, activators)from their respective sources to the single reaction cell and thefurther delivery of products and by-products (peptides, waste, cleavedresin) from the cell to their respective destinations. It will beunderstood that the particular flow paths and valve locationsillustrate, rather than limit, the present invention.

As noted earlier, the microwave instrument portions of the synthesisinstrument can essentially be the same as those set forth in a number ofcommonly assigned and co-pending U.S. patent applications. Accordingly,FIG. 7 is included to highlight certain aspects of the microwave portionof the instrument without overly burdening the specification herein. Inparticular, FIG. 7 is essentially the same as FIG. 1 in previouslyincorporated application Ser. No. 10/064,261 filed Jun. 26, 2002. FIG. 7illustrates a microwave cavity 117 shown in cutaway fashion for clarity.The cavity is attached to a wave guide 120, which is in microwavecommunication with an appropriate source (not shown). Microwave sourcesare widely available and well understood by those of ordinary skill inthis art, and include magnetrons, klystrons, and solid state diodes.FIG. 7 illustrates a test tube-shaped cell 121 in the cavity 117 andsuch can be used if desired for the reactions of the present invention,although the pear-shaped vessel 45 is generally preferred.

In order to carry out the simultaneous cooling, the instrument includesa cooling gas source (not shown) which delivers the cooling gas to theinlet fitting 122 on the flow valve 123 (typically a solenoid). Duringactive cooling, the solenoid 123, which is typically softwarecontrolled, directs cooling gas through the tubing 124 and to thecooling nozzle 125, which directs the cooling gas on to the reactionvessel 121.

It should be pointed out, however, that other cooling mechanisms may beadapted to this method, such as a stream of refrigerated air or a liquidcooling mechanism that circulates refrigerated liquid around thereaction cell in a manner that would not interfere with the transfer ofmicrowave energy.

FIG. 7 also illustrates a cylindrical opening 126, which is typicallyused to permit temperature observation of the reaction vial 121. Suchtemperature observation can be carried out with any appropriate device,which can normally include a fiber optic device of the type that canmeasure the temperature of an object by reading the infrared radiationproduced by the object. Such devices are well understood in the art, andwill not be discussed in further detail herein, some aspects havingalready been discussed in the incorporated references.

In preferred embodiments, the microwave source is capable of, but notlimited to, “spiking” microwave energy. In other words, the microwavesource is capable of generating high power for a short length of time asopposed, but not limited to, low power for a longer period of time. Thisfeature aids in preventing the undesirable effect of overheating thecontents of the reaction vessel and appears to increase the rate ofreaction as well.

The apparatus optionally includes an infrared photosensor for measuringtemperature. The infrared sensor does not contact the reaction cellcontents, yet still accurately measures the average temperature of thereaction cell contents and not merely the air temperature surroundingthe contents. Infrared temperature analysis is more accurate,non-intrusive, and allows for a more simplified apparatus designcompared to a probe or the like, which measures only a localized areaand would require physical contact of the contents.

The second passageway is further characterized by a filter whichprevents the passage of resin. Additionally, the first and secondpassageways are in fluid communication with each other with respect tothe movement of liquid solvents and flowing solids; herein the term“flowing solids” refers to resin, with or without amino acids orpeptides attached, and suspended in an appropriate solvent.

In another aspect, the invention is a method for the solid phasesynthesis of one or more peptides that incorporates the use of microwaveenergy. Microwave energy applied to the contents of the reaction cellduring the deprotecting, activating, coupling, and cleaving stepsgreatly decreases the length of time necessary to complete thesereactions. The method for applying microwave energy may be moderated bythe microwave source in such a way as to provide the fastest reactiontime while accumulating the least amount of heat, thus more microwaveenergy may be applied and heat-associated degradation of the reactioncell contents does not occur. This method includes, but is not limitedto, spiking the microwave energy in large amounts for short lengths oftime.

The method optionally includes the synthesis of a complete peptide oftwo or more amino acids in a single reaction vessel, and may include thecoupling of one or more amino acids to one or more amino acids that areattached to the solid phase resin.

The method includes cooling the reaction cell, and thus its contents,during and between applications of microwave energy up to and includingthe final cleaving step. The cooling mechanism of the method operatesduring amino acid extension cycles, the term “cycle” used herein torefer to the deprotection, activation, and coupling necessary to linkone amino acid to another. The cooling system can also operate duringand between applications of microwave energy in a given cycle to keepthe bulk temperature of the reaction cell contents down. The coolingsystem can also operate when the complete peptide is cleaved from theresin.

Alternatively, it has also been discovered that controlling the power,rather than strictly controlling the temperature, can also provide adesired control over the progress of a reaction. As noted elsewhereherein, the use of a variable or switching power supply can help servethis purpose, an example of which is given in commonly assigned U.S.Pat. No. 6,288,379; the contents of which are incorporated entirelyherein by reference.

The method includes agitating the contents of the reaction cell withnitrogen gas in order to promote maximal exposure of the resin and anyattached amino acids or peptides to solvents and free amino acids.

In a preferred embodiment, the method comprises transferring a firstcommon amino acid linked to a resin of choice, both suspended in anappropriate solvent, to the reaction cell via pressurized nitrogen gas.A deprotection solution is then pumped into the reaction cell. Thisprocess is accelerated by the application of microwave energy, and theheat generated by the microwave energy is minimized by a coolingmechanism. Multiple deprotection steps may be executed. The deprotectionsolution is then withdrawn from the reaction cell, leaving thedeprotected, common amino acid linked to the resin. After several (threeto five) resin washes of approximately one resin volume each using anappropriate solvent and removing the wash solvent, the next “free”common amino acid or acids (dissolved in solution) is added to thereaction cell along with an activating solution. The activation of thefree amino acid is accelerated by the application of microwave energy,and the reaction cell temperature is controlled by a cooling mechanismas described above. The method further comprises coupling the free aminoacid or acids to the deprotected, linked amino acid, forming a peptide,using microwave energy to accelerate the method. As above, heatgenerated by the microwave energy is minimized by a cooling mechanism.The coupling step is further preferred to include nitrogen agitation ofthe reaction cell contents. Completion of this step represents one cycleof one or more amino acid addition. Following the coupling step, theactivation solution is withdrawn and the resin is washed as above. Thecycle is repeated until the desired peptide sequence is synthesized.Upon completion of peptide synthesis, a further deprotection step may becarried out to remove protective chemical groups attached to the sidechains of the amino acids. This deprotection step is carried out asdescribed above. The resin containing the attached, completed peptide isthen washed as above with a secondary solvent to prepare the peptide forcleavage from the resin. Following the removal of the secondary solvent,cleaving solution is added to the reaction cell and cleaving isaccelerated by the application of microwave energy, and the heatgenerated by the microwave energy is minimized by a cooling mechanism.Upon completion of cleaving, the peptide product is transferred to aproduct tube. Optionally, the peptide may be “capped” at any pointduring the synthesis process. Capping is useful to terminateincompletely coupled peptides, assist in proper folding of the peptidesequence, and to provide a chemical identification tag specific to agiven peptide. However, these modifications decrease the solubility ofsynthetic peptides and thus must be carefully considered. Capping iscarried out for example, but not limited to, using acetic anhydride orfluorous capping in solid phase synthesis, or by attaching any of alarge variety of chemical groups such as biotin to either theN-terminal, C-terminal or side chain of a peptide.

In another embodiment, the invention comprises de-protecting first aminoacid linked to a solid phase resin by removing protective first chemicalgroups, activating chemical groups on a second amino acid to prepare thesecond amino acid for coupling with the first amino acid, coupling theactivated the second amino acid to the de-protected first amino acid toform a peptide from the first and second amino acids, cleaving thepeptide from the solid phase resin, applying microwave energy toaccelerate the de-protected, activating and coupling cycle, and applyingmicrowave energy to accelerate the cleaving step.

It is, of course, the usual procedure to add a number of amino acids toone another to form a peptide sequence. Accordingly, the method can, andusually, comprises repeating the de-protecting, activating and couplingcycle to add third and successive acids to form a peptide of a desiredsequence.

In that regard, it will be understood that as used herein, terms such as“first,” “second,” or “third” are used in a relative rather thanabsolute sense.

In a particularly preferred embodiment, the method comprisessuccessively de-protecting, activating and coupling a plurality of aminoacids into a peptide in a single vessel without removing the peptidefrom the solid phase resin between the cycles. This, and additionalaspects, of the invention will be understood with regard to thediscussion of the figures.

In another embodiment, the method comprises proactively cooling thevessel and its contents during the application of microwave energy tothereby prevent undesired degradation of the peptide or acids bylimiting heat accumulation that would otherwise result from theapplication of the microwave energy.

As is typical in peptide synthesis, the de-protecting step comprisesde-protecting the alpha-amino group of the amino acid, but can alsocomprise de-protecting side chains on the amino acids of the peptide,both under the microwave and radiation. Similarly, the activating steptypically comprises activating the alpha-carboxyl group of the secondamino acids.

Because the amino acids and peptides are sensitive to excessive heat,and in addition to the proactive cooling step just described, the stepof applying the microwave energy can comprise “spiking” the applicationof microwave energy to relatively short-time intervals to therebyprevent undesired degradation of the peptidal acids by limiting heataccumulation that could be encouraged by the continuous application ofthe microwave energy. As used herein, the term “spiking” refers to thelimitation of the application of microwave energy to the relative shorttime intervals. Alternatively, the microwave power can be supplied froma switching power supply as set forth in commonly assigned U.S. Pat. No.6,288,379, the contents of which are incorporated entirely herein byreference.

In other embodiments, the peptide synthesis process can compriseactivating and coupling in situ using a carbodiimide type coupling freeagent.

In another aspect, the invention is a process for accelerating the solidphase synthesis of peptides. In this aspect, the method comprisesdeprotecting a protected first amino acid linked to a solid phase resinby admixing the protective linked acid with a deprotecting solution in amicrowave, transparent vessel while irradiating the admixed acid andsolution with microwaves, and while cooling the admixture (oralternatively controlling the applied power, or both) to prevent heataccumulation from the microwave energy from degrading the solid phasesupport or any of the admixed compositions. In particular, the methodcomprises deprotecting the alpha-amino group of the amino acid, and mosttypically with a composition suitable for removing protective chemicalsselected from the group consisting of fmoc and boc. As is known to thosefamiliar with this chemistry, the deprotecting step can also comprisedeprotecting the side chain of the amino acid. In those circumstances,the deprotecting step comprises using a composition suitable forremoving t-butyl-based side chain protecting groups.

Following the deprotecting step, the method comprises activating asecond amino acid by adding this second amino acid and an activatingsolution to the same vessel while irradiating the vessel with microwavesand while simultaneously cooling the vessel to prevent heat accumulationfrom the microwave energy from degrading the solid face support or anyof the admixed compositions.

The method next comprises coupling the second amino acid to the firstacid while irradiating the composition in the same vessel withmicrowaves, and while cooling the admixture to prevent heat accumulationfrom the microwave energy from degrading the solid phase support or anyof the admixed compositions.

Finally, the method comprises the step of cleaving the linked peptidefrom the solid phase resin by admixing the linked peptide with acleaving composition in the same vessel while irradiating thecomposition with microwaves, and while cooling the vessel to preventheat accumulation from the microwave energy from degrading the solidphase support or the peptide.

The activating step can also comprise activating and coupling the secondamino acid using an in situ activation method and composition such asphosphorium or uranium activators, HATU, HBTU, PyBOP, PyAOP, and HOBT.

Once again, because the synthesis of peptides almost always includes theaddition of three or more acids into the chain, the method can comprisecyclically repeating the steps of deprotecting, activating and couplingfor three or more amino acids in succession to thereby synthesize adesired peptide.

In a particular embodiment of the invention, the successive steps ofdeprotecting, activating, coupling and pleading are carried out in thesingle reaction vessel without removing the peptide from the solid phaseresin or from the vessel between cycles.

The method can further comprise agitating the admixture, preferably withnitrogen gas during one or more of the deprotecting, activating,coupling and pleading steps. Any gas can be used for the agitation,provided it does not otherwise interfere with the synthesis chemistry,the peptides or the amino acids, but nitrogen is typically preferred forthis purpose because of its wide availability, low cost and chemicalinertness with respect to the particular reactions.

EXPERIMENTAL

Peptide: Asn-Gly-Val

MW=288

Scale=0.10 mmol

Resin used=Fmoc-Val-Wang Resin

Resin substitution=0.27×10-3 moles/gram resin

Microwave Protocol:

For all reactions in this peptide the microwave power was initially setat 50 W then regulated to maintain the temperature below 60° C.

Deprotection: A 20% Piperidine in DMF solution was used fordeprotection. The reaction was performed for 30 seconds in microwave,and then repeated with new deprotection solution for 1:00 minute inmicrowave.

Coupling: Activation was performed with 0.40 mmol HCTU, 0.80 mmol DIPEA,and 0.40 mmol of each Fmoc-amino acid for each coupling in thesynthesis. Approximately 10 mL of DMF was used to dissolve the mixture.The reaction was performed for 5:00 min. in the microwave.

Washing: The vessel was filled with approximately 10 mL of DMF andrinsed 5 times after each deprotection and coupling step.

Cleavage: Cleavage was performed with 95% TFA and 5% H2O for 90:00 min.

Peptide was precipitated in 50 mL of cold ethyl ether overnight. Productwas collected and dried. Mass Spectrum was obtained of crude productfrom electrospray ionization mass spectrometry using a ThermoFinniganAdvantage LC/MS.

Results: The Electrospray Ionization Mass Spectrum (FIG. 8) showed asingle peak at 289.1 corresponding to the MW of Asn-Gly-Val. No otherpeaks corresponding to incomplete couplings were observed.

Peptide: Gly-Asn-Ile-Tyr-Asp-Ile-Ala-Ala-Gln-Val (SEQ ID NO: 1)

MW=1062

Scale=0.25 mmol

Resin used: Fmoc-Val-Wang Resin

Resin substitution=0.27×10-3 moles/gram resin

Microwave Protocol:

This peptide was synthesized with a power time control method.

Deprotection: A 20% Piperidine in DMF solution was used fordeprotection. The deprotection was performed with 25 W of microwavepower for 30 seconds, and then repeated with new deprotection solutionfor 1:00 min. in the microwave.

Coupling: Activation was performed with 0.9/1.0 mmol of HBTU/HOBtrespectively, 2 mmol of DIPEA, and 1.0 mmol of Fmoc-amino acid for eachcoupling in the synthesis. Approximately 15 mL of DMF was used todissolve the mixture. The coupling reaction was done in 5:00 min. in themicrowave with power alternating between on for 15 seconds and off for45 seconds. The first cycle of power was 25 W, and the remaining fourwere each 20 W.

Washing: The vessel was filled with approximately 15 mL of DMF andrinsed 5 times after each deprotection and coupling step.

Cleavage: Cleavage was performed with 95% TFA, 2.5% H2O, and 2.5% TIS.

Peptide was precipitated in 100 mL of cold ethyl ether overnight.Product was collected and dried. Mass Spectrum was obtained of crudeproduct from electrospray ionization mass spectrometry using aThermoFinnigan Advantage LC/MS.

Results: The Electrospray ionization mass spectrum (FIG. 9) shows a peakat 1063.3 that corresponds to the desired peptide mass. No peaks weredetected for incomplete couplings. A second peak was obtained at 1176.5that corresponds to the desired peptide with an extra Ile amino acid.This corresponds to incomplete removal of the deprotection solutionbefore one of the Ile coupling reactions and allowing two Ile aminoacids to be added to the peptide.

In the drawings and specification there have been disclosed typicalembodiments of the invention. The use of specific terms is employed in adescriptive sense only, and these terms are not meant to limit the scopeof the invention being set forth in the following claims.

1. A process for the solid phase synthesis of peptides, which comprises:(a) transferring solid phase resin particles between a resin sourceexternal to a single microwave transparent vessel and into the microwavetransparent vessel; (b) deprotecting a first amino acid linked to a thesolid phase resin particles by removing protective chemical groups fromthe first acid; (c) activating chemical groups on a second amino acid toprepare the second amino acid for coupling with the first amino acid;(d) coupling the activated second amino acid to the deprotected firstamino acid to form a peptide from the first and second amino acids; (e)accelerating at least the deprotecting and coupling steps by applyingmicrowave energy during the deprotecting, and coupling steps; and (f)successively deprotecting, activating, and coupling a plurality of aminoacids into a peptide in the single microwave transparent vessel withoutremoving the peptide from the single vessel between cycles.
 2. A processaccording to claim 1 comprising cleaving the peptide from the solidphase resin particles while applying microwave energy to accelerate thecleaving step.
 3. A process according to claim 1 comprising transferringthe solid phase resin particles as a suspension between the resinparticle source and the microwave transparent vessel.
 4. A processaccording to claim 1 comprising transferring the solid phase resinparticles into the microwave transparent vessel and then solvating theresin particles.
 5. A process according to claim 1 comprisingtransferring the solid phase resin particles as a flowing solid into themicrowave transparent vessel.
 6. A peptide synthesis process accordingto claim 1, comprising successively deprotecting, activating, andcoupling a plurality of amino acids into a peptide in the single vesselwithout removing the peptide from the solid phase resin particlesbetween cycles.
 7. A peptide synthesis process according to claim 1,comprising proactively cooling the vessel and its contents during theapplication of microwave energy to thereby prevent undesired degradationof the peptide or acids by limiting heat accumulation that wouldotherwise result from the application of the microwave energy.
 8. Apeptide synthesis process according to claim 1, wherein the deprotectingstep comprises deprotecting the alpha-amino group of the amino acid. 9.A peptide synthesis process according to claim 1, further comprisingdeprotecting side chains on the amino acids of the peptide undermicrowave irradiation.
 10. A peptide synthesis process according toclaim 1, wherein the activating step comprises activating thealpha-carboxyl group of the second amino acid.
 11. A peptide synthesisprocess according to claim 1, wherein the step of applying the microwaveenergy comprises limiting the application of microwave energy torelatively short time intervals to thereby prevent undesired degradationof the peptide or acids by limiting heat accumulation that could beencouraged by the continuous application of the microwave energy.
 12. Apeptide synthesis process according to claim 1 further comprisingactivating and coupling using a in situ method and a compositionselected from the group consisting of phosphorium activators, uroniumactivators, HATU, HBTU, PyBOP, PyAOP, and HOBT.
 13. A peptide synthesesprocess according to claim 1 comprising monitoring the temperature ofthe vessel and moderating the applied power based upon the monitoredtemperature.
 14. A peptide synthesis process according to claim 1comprising moderating the applied power based on the status of thereaction.